Biomimetic electrospun PVDF/self-assembling peptide piezoelectric scaffolds for neural stem cell transplantation in neural tissue engineering

Piezoelectric materials can provide in situ electrical stimulation without external chemical or physical support, opening new frontiers for future bioelectric therapies. Polyvinylidene fluoride (PVDF) possesses piezoelectricity and biocompatibility, making it an electroactive biomaterial capable of enhancing bioactivity through instantaneous electrical stimulation, which indicates significant potential in tissue engineering. In this study, we developed electroactive and biomimetic scaffolds made of electrospun PVDF and self-assembling peptides (SAPs) to enhance stem cell transplantation for spinal cord injury regeneration. We investigated the morphology and crystalline polymorphs of the electrospun scaffolds. Morphological studies demonstrated the benefit of using mixed sodium dodecyl sulfate (SDS) and SAPs as additives to form thinner, uniform, and defect-free fibers. Regarding electroactive phases, β and γ phases—evidence of electroactivity—were predominant in aligned scaffolds and scaffolds modified with SDS and SAPs. In vitro studies showed that neural stem cells (NSCs) seeded on electrospun PVDF with additives exhibited desirable proliferation and differentiation compared to the gold standard. Furthermore, the orientation of the fibers influenced scaffold topography, resulting in a higher degree of cell orientation in fiber-aligned scaffolds compared to randomly oriented ones.


Introduction
Spinal cord injuries (SCIs), the leading cause of irreversible paralysis, have a profound and devastating impact on the lives of more than 20 million people worldwide due to the limited capability to regenerate or replace neuronal tissue. 1 Previous studies in the eld of neurobiology revealed that the deciency of neuronal regeneration in the central nervous system (CNS) is not only because of the intrinsic lack of CNS axon regeneration ability but also because of extrinsic cell growth inhibition signals conferred by the CNS environment within the damaged tissue. 2 Thus, if neurons are provided with the correct set of biological and physical stimuli, regeneration of the damaged neuronal tissue should be favored.Neural regeneration using Neural Stem Cells (NSCs) has demonstrated tremendous potential for neuro-regenerative therapies in humans by promoting angiogenesis and neurogenesis. 35][6][7] Transplanted cells and regenerating nervous bers require pro-regenerative substrates, favoring their engrament and spatially guiding the host tissue regeneration, to regenerate the neural circuitry preexisting the injury.Advances in biomaterial design, manufacturing, and surface chemistry have vastly improved the safety and function range of implantable biomaterials such as cell scaffolding.Despite signicant progress in neural regeneration on electrospun polymer scaffolds containing conventional neurotrophic factors such as nerve growth factor (NGF), the short half-life time and fast diffusion of factors like NGF signicantly limit those scaffolds' efficiency in vivo. 8Electrical stimulation (ES) has been demonstrated to be a promising alternative to conventional growth factor treatments for the differentiation of various cell types, including neurons.This differentiation is mediated through multiple signaling pathways, including the MAPK/ERK pathway and the cAMPdependent pathway. 9,10The nervous system (NS) is highly inuenced by ES, which serves as the primary means of communication. 11Applying ES to scaffolds serves to mimic not only the electrical properties of the NS but also to reduce inammatory response aer implantation. 12Moreover, ES application improves the promotion of neural cells migration, proliferation, and differentiation, aiding the neurite extension process and resulting in a larger mean number of axons and an increased number of blood vessels in endoneurial and nervous areas. 13,14iezoelectric materials, which can provide in situ ES without external chemical or physical support, open new frontiers for future bioelectric therapies.The native mechanisms of neuronal activation by ES are described by voltage-gated ion channels that ES causes both membrane potential depolarization and ion ow across the membrane via voltage-gated channels. 11olyvinylidene uoride (PVDF) serves as an electroactive biomaterial with the ability to enhance bioactivity through instantaneous ES.It can be tailored to create intelligent scaffolds that stimulate and regulate cell growth and behavior 15 possesses piezoelectricity and processability, indicating significant potential in tissue engineering. 16This attribute is particularly advantageous in neural tissue engineering, enabling the development of scaffolds responsive to electrical cues, mimicking the native neural environment.Such electrical responsiveness plays a crucial role in inuencing cell behavior and promoting neural regeneration. 17lectrospun brous PVDF scaffolds can effectively mimic the structure and components of the targeted extracellular matrix (ECM), both biochemically and electrically.This makes them promising scaffolds for neural tissue engineering.The high surface area and interconnected porous structure of these scaffolds create an environment conducive to cell adhesion, proliferation, and migration. 14he mechanical properties of electrospun PVDF scaffolds can be customized to match those of native neural tissue.This customization is essential for providing the necessary support and mechanical cues to cells during tissue regeneration.PVDF's exibility and durability make it suitable for replicating the mechanical properties of neural tissue. 14dditionally, PVDF, as a hydrophobic polymer, can be modied or combined with other biomaterials to enhance its hydrophilicity, biochemical properties, and compatibility with neural tissue.Surface modications, such as the incorporation of bioactive molecules or peptides, can promote specic cellular interactions and improve neural tissue regeneration.Preliminary in vitro studies on electrospun PVDF and PVDF-TrFE scaffolds have demonstrated the promotion of neural differentiation in PC12 cells compared to conventional in vitro differentiation protocols using NGF. 18Furthermore, hNSCs showed better differentiation into b-III tubulin (bIII-TUB)-positive cells and greater average neurite length, 19 especially with low-aligned PVDF scaffolds. 20These scaffolds also facilitated the alignment and proliferation of Schwann cells and broblasts. 21,22PVDF membrane was also used as an articial nerve conduit for peripheral nerve injury repair.The PVDF scaffold revealed a sufficient scaffold biocompatibility with Schwann cells and no apparent cytotoxicity regarding neonatal rat and adult human Schwann cells along with enhanced bidirectional outgrowth of axons. 23On the other hand, fabricating a piezoelectric PVDF/graphene oxide scaffold via a non-solventinduced phase separation method for nerve tissue engineering applications resulted in the improvement of attachment, spreading, and proliferation of PC12 cells. 24n this study, modied PVDF scaffolds with self-assembling peptides (SAPs) were manufactured to biomimic the microenvironment of the ECM for NSC growth and increase biodegradation by manipulating the scaffold surface's hydrophilicity which downregulates inammatory response and promotes anti-inammatory M2 macrophage growth. 25Biofunctionalized SAPs have been extensively investigated as NSCs transplantation carriers in neural tissue engineering and are appreciated for their excellent biocompatibility and biodegradability properties. 26,27On this basis, we explored the inuence of solution parameters (co-solvent) and additives (SDS and SAPs) on the resulting PVDF nanobers in terms of morphology, quantities of polymorph phases, and direct piezoelectric effect due to sound-induced strain rates at various sound frequencies.We investigated the role of the piezoelectric PVDF and SAPs as parameters benecial to a pro-regenerative micro-environment by measuring the cell viability, differentiation, proliferation, and by the studying adhesion patterns of NSCs on the tested scaffolds.

Materials and preparation
2.1.1Materials.Polyvinylidene uoride (PVDF), with an average molecular weight of 275 000 Mw, dimethylformamide (DMF) (solvent), acetone (co-solvent), and sodium dodecyl sulfate (SDS) (anionic surfactant) were purchased from Merck (Merck Millipore, Darmstadt, Germany), Sigma Aldrich (Sigma Aldrich Chemie GmbH, München, Germany) and used as received.The linear sequence NH 2 -FAQRVPPGGG(LDLK) 3 -CONH 2 peptide (dubbed FAQ(LDLK) 3 ) was synthesized via microwave-assisted Fmoc SPPS on a 0.56 mmol g −1 Rink Amide 4-methylbenzhydrylamine resin (0.5 mmol g −1 substitution) using a CEM Liberty Blue system (CEM Corp., Matthews, NC, Canada) with a 0.25 mmol scale. 26.1.2Preparation of the scaffolds using electrospinning.Electrospinning solutions for PVDF-based bers were prepared by dissolving 25% w/v of PVDF in either pure solvent, DMF, or containing acetone with different ratios (100 : 0 and 60 : 40 v/v, DMF to acetone).The PVDF was rst dissolved in the chosen primary solvent (DMF) via continuous stirring for 4 h at 70 °C.Aer cooling, acetone was added (if required), and the solution was stirred again for 2 h at room temperature.In the case of the solutions that included SDS, the concentrations of PVDF and SDS were 24.75% w/v and 0.25% w/v, respectively.In the case of the solutions that include only the peptide FAQ(LDLK) 3 , the concentrations of PVDF and FAQ(LDLK) 3 were 23.75% w/v and 1.25% w/v, respectively.Finally, in the case of the solution which includes both SDS and FAQ(LDLK) 3 peptide, the concentrations of PVDF, SDS, and FAQ(LDLK) 3 were 23.5% w/v, 0.25% w/v, and 1.25% w/v, each.Table 1 summarizes the preparation conditions of the solutions and the electrospinning conditions used to produce the bers.
The bers were electrospun using Electroris (FNM Ltd., Fanavaran Nano-Meghyas Company, Tehran, Iran,), an electrospinning device with humidity and temperature controllers.The solutions were loaded in a syringe (diameter d = 8.7 mm, Terumo) and placed in the horizontal direction.A Gamma highvoltage research power supply (ES50P-10W, Gamma High Voltage Research, Inc., Ormond Beach, FL, USA) was used to charge the solution in the syringe with a positive DC voltage.The positive electrode was connected to the 22G needle (diameter d = 0.7 mm) of the syringe and the ground electrode was attached to the collector.A controllable syringe pump (Harvard Apparatus Model 44 Programmable Syringe Pump) in the range of 0.01-100 ml h −1 was used to feed the needle.Random and aligned bers were collected on a at target and rotating drum covered by aluminum, respectively.The applied parameters were voltage tension = 17/18 kV, tip-collector distance = 20 cm, ow rate = 200/400 ml h −1 , humidity = 30%, temperature = 22 °C, and rotating rate = 2000 rpm (for aligned bers).

Scanning electron microscopy (SEM)
. SEM imaging was conducted with a Tescan VEGA TS 5136XM (TESCAN Company, Brno, Czech Republic) to investigate the samples' morphology.All samples were sputter-coated with a nominally 10 nm thin gold lm using a Quorum Tech Q150R S (Quorum Company, East Sussex, UK) sputter coater.The ber diameters, brous and bead contents, and directionality were measured using ImageJ 1.52a. 28,29.2.2 Porosity.Pore size was measured by ImageJ 1.52a.The apparent density and porosity of electrospun brous mats were calculated using the following equations, where the thickness of the brous mats was measured by SEM: Nanofiber mat apparant density À g cm À3 Á ¼ Nanofiber mat mass ðgÞ Nanofiber mat thickness ðcmÞ Â Nanofiber mat area ðcm 2 Þ (1) 2.2.3 Fourier transform infrared spectroscopy (FTIR).FTIR spectra were recorded on a PerkinElmer FTIR spectrometer (PerkinElmer, Waltham, MA, USA) in the spectral range 400-4000 cm −1 with a resolution of 1 cm −1 in transmission mode.The FTIR measurements were repeated three times at random locations for each scaffold type to minimize error.To minimize the interference of the solvents' peaks, entire samples were vacuum-dried overnight.Data processing was performed using Origin 2020 soware (OriginLab Corporation, Northampton, MA, USA).All the measured spectra are background-corrected and normalized.A peak analyzer was used to perform nonlinear tting of the peaks in the spectral data.Baseline corrections were performed using a second derivative (zeroes) method to nd anchor points and detect the baseline.Hidden peaks were also detected in the spectral range 700-925 cm −1 and 925-1350 cm −1 by a second derivative method followed by smoothing with the ten-point Savitsky-Golay function with 2nd order polynomial.The deconvoluted spectral peaks were tted with the Gaussian function.The positions and the number of the components (used as an input le for the curve-tting function) were obtained from both the second derivative and the deconvoluted spectra.The quality of the tting was estimated by standard deviation.
Since the peak at 840 cm −1 can be assigned to the b, g, or both phases and 763 cm −1 is attributed to the a phase, the relative fraction of the electroactive b and g phases (F EA ) in terms of crystalline components in any samples can be quan-tied with the following equation: 30 where, I 840 and I 763 are the absorbencies at 840 and 763 cm −1 respectively; K 840 and K 763 are the absorption coefficients at the respective wave numbers, whose values are 7.7 × 10 4 and 6.1 × 10 4 cm 2 mol −1 , respectively.Note that this equation implies the sample is composed only of a and b phases and that the g phase resonance also overlaps the b phase in this region.The quan-tication of individual b and g phases can be performed by using the absorbance of the two bands 1275 and 1234 cm −1 .However, a much more reliable method is proposed by calculating the peak-to-valley height ratio between the peaks around 1275 and 1234 cm −1 and their nearest valley, as illustrated in the equations below 30 where, DH b and DH g are the height differences between the peak at ∼1275 cm −1 and the nearest valley at ∼1260 cm −1 , and the peak at ∼1234 cm −1 and the nearest valley at ∼1225 cm −1 , respectively.In summary, with FTIR we can determine the relative proportion of a, b and g phases. 30.2.4 Differential scanning calorimetry (DSC).DSC data were recorded on a STARe DSC analysis system (Mettler Toledo) equipped with low-temperature apparatus and calibrated with high-purity indium.The experiments were run under a nitrogen atmosphere in standard 40 mL Al pans.DSC measurements were performed between −80 °C and 300 °C at 10 °C min −1 on samples having masses of about 5 mg.The degree of crystallinity (X c ) was estimated by using a melting enthalpy (DH°) of 104.6 J g −1 for a 100% crystalline PVDF.31 2.2.5 Water contact angle.The contact angle analysis was performed using an in-house contact angle setup, consisting of a camera (Fastcam Nova S6, Photron) with Tokina AT-X PRO D (100 mm F2.8 MACRO) as an optical lens and backlight illumination.Contact angles were measured by dispensing water with a syringe pump (Harvard Apparatus, Pump 11 Pico Plus Elite) at a rate of 10 ml min −1 , with drop volumes in the range of 5-10 ml.Obtained images and videos were analyzed with ImageJ 1.52a.
2.2.6 Piezoelectric response test.To measure the voltage generated upon sound stimulation, the electrospun membranes with sizes of 2.5 cm × 4 cm were sandwiched between two aluminum foils as an electrode.A copper wire was attached to the electrode on each side to provide a connection with a multimeter (Fluke® 175 True-RMS Digital Multimeter).The membranes were covered with a thin layer of transparent Polyethylene Terephthalate (PET).To demonstrate the piezoelectricity of PVDF-based membranes, the degrees of sample electrical response were qualitatively measured as the samples were stimulated with different sound frequencies from 1 to 2000 Hz.The piezoelectric output was measured as the resulting voltage over frequency.Controls were performed by measuring the voltage with no external force applied.Fig. 1 shows the schematic of the piezoelectric response test.
2.2.7 Culturing murine neural stem cells (mNSCs).mNSCs were isolated from the striatum of 8-week-old CD1 albino mice, specically from the Sub-Ventricular Zone (SVZ).The hNSCs were cultured in T75 asks at a density of 1 × 10 4 cells per cm 2 , utilizing a serum-free medium supplemented with basic broblast growth factor (10 ng per ml bFGF) and epidermal growth factor (20 ng per ml EGF).The cells were maintained in a humidied incubator at 37 °C, 5% O 2 , and 5% CO 2 .Once the neurospheres reached a diameter of 100-150 mm, they were mechanically dissociated by pipetting to transition to a singlecell state.

2.2.8
In vitro cellular differentiation.Cells were cultured on diverse electrospun scaffolds, which were pre-attached to the bottom of a 48-well plate (Corning® Costar® TC-Treated Multiple Well Plates).The cell density was set at 3 × 10 4 cells per cm 2 in a fresh basal medium supplemented solely with FGF (10 ng ml −1 ).For neuronal and glial differentiation, aer 2 days in vitro (DIV), the medium was gradually substituted with a medium containing leukemia inhibitory factor (LIF, 20 ng ml −1 ) and brain-derived neurotrophic factor (BDNF, 20 ng ml −1 ) to facilitate the differentiation of cells toward three neuronal and glial populations in hNSC progeny.
As a positive control, CULTREX-BME® (R&D Systems) was utilized.It was positioned in wells devoid of scaffolds and incubated overnight.Following this, the Cultrex was aspirated and substituted with bFGF media.
2.2.8.1 Cell viability.Aer 7 DIV of differentiation, cell proliferation was assessed via CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS assay, Promega, Madison, WI, USA): MTS solution was added to the culture media (1 : 5) and incubated for 1 h at 37 °C.The supernatant of each sample was quantied via Innite M200 PRO plate reader (Tecan, Männedorf, Switzerland) by measuring absorbance at 490 nm.
2.2.9 Immunouorescence.Aer 7 DIV, cells were xed using paraformaldehyde (PFA, Sigma-Aldrich 95%) following this protocol: a 5 minute wash in PBS, xation with 2% PFA for 10 minutes, followed by an additional xation with 4% PFA for 10 minutes.The PFA solutions were diluted in PBS.Aer two 5 minute washes in PBS, cell membranes were permeabilized by treating the cells with 0.3% Triton X-100 (Sigma-Aldrich) for 10 minutes at 4 °C.To block nonspecic binding sites, cells were then exposed to 10% normal goat serum (NGS, GIBCO) for 1 h at room temperature.Aer three washes in PBS, primary antibodies were diluted in a buffer composed of PBS, 1% NGS, and 0.3% triton and applied overnight at 4 °C.Specically, Glial Fibrillary Acidic Protein (GFAP) was used to label astrocytes, bIII-Tubulin (bIII-TUB) for neurons, and Galactocerebroside (GALC) along with the oligodendrocyte marker (O4) for oligodendrocytes.Following three 5 minutes of washes in PBS each, secondary antibodies were diluted in the same buffer used for the primary antibodies and applied for 2 h at room temperature in the dark.Subsequently, the cells were again washed in PBS three times for 5 minutes each.Hoechst (diluted at 1 : 500 in PBS) was then applied for 10 minutes in the dark to label cell nuclei.Aer a 5 minute wash in PBS and Milli-Q water, Fluo-rSave™ (Millipore) reagent was applied to preserve immuno-uorescence.It's noteworthy that solutions for GALC-O4 were prepared without 0.3% Triton.Both assays were conducted in triplicate.A minimum of three randomly selected elds for each independent experiment were imaged at 20× magnication using a Zeiss Microscope with Apotome System for staining.Cell quantication was performed by manually counting positive cells for each marker using NIH-Fiji soware.Additionally, measurements were taken for the length of axons and the soma area of astrocytes.Colored images were transformed into binary images (black and white, 8-bit), and adjustments were made using the automated threshold algorithm with threshold values ranging from 0 to 255.Aer isolating a single cell, the scale was established, the perimeter traced, and the soware automatically computed the dened area.Following this, the length of the axons was measured by setting the scale and tracing the axon.An additional valuable tool employed was the 'Directionality' automatic orientation calculator, which provided the frequency distribution of each orientation.
2.2.10 Statistical analysis.Data was processed using Excel, GraphPad Prism 8, and OriginPro 1.52a soware.Reported values are as means ± standard error of the mean (SEM).All experiments were repeated three times.Secondary structures were analyzed using One-way ANOVA (paired comparison plot), and Tukey's post hoc test was used for comparative analysis and statistical signicance, delineated as *p # 0.05, **p # 0.01, and ***p # 0.001.The MTS assay was processed through one-way ANOVA followed by Dunnett's multiple comparison tests.For in vitro studies, the bIII-TUB and GFAP were evaluated by twoway ANOVA followed by Bonferroni's multiple comparison test, and GalC-O4 was performed via one-way ANOVA followed by Dunnett post-test.The axon's length and soma's area were evaluated by ordinary one-way ANOVA followed by Tukey's multiple comparison test.

Results and discussion
Initially, the physical characteristics of PVDF scaffolds were investigated through a suitable tissue engineering substrate lens.With insight from previous studies, the correlation of ber morphology (brous and bead content), orientation, and additives on the electroactive phase content of electrospun PVDF mesh were examined, along with cell survival and differentiation. 30They demonstrated that a lower bead content and the most uniform medium-range ber thickness, consequently resulting in the highest content of the electroactive phase and ber alignment, have a profound effect on neuronal cell alignment. 301 Characterization of ber morphology, porosity, directionality, and contact angle Random and aligned scaffolds were fabricated by electrospinning 25% (w/v) PVDF in DMF:acetone (60 : 40 and 100 : 0) solutions with and without SDS and FAQ(LDLK) 3 .All scaffolds excluding those lacking additive and co-solvents exhibited uniform and defect-free bers with a mean diameter of 200-600 nm, a necessity for the formation and improvement of electroactive phase content as Ico et al. showed the negative logarithmic relationship between ber diameter and piezoelectric constant.32 On the other hand, a signicant reduction in bead content (including greater a phase) for scaffolds comprising SDS and FAQ(LDLK) 3 leads to higher electroactivity.This was shown in our previous study that a and b phases are directly related to specic ber morphologies and that they make up the dominant fraction of bead and brous parts of the bers.30 Regarding the scaffolds modied by SAPs, it was revealed that the addition of SAPs can signicantly reduce both the bead content and the average ber diameter (Fig. 2).Therefore, these ber diameters and morphological patterns may prove ideal both from a purely physical perspective and in the context of piezoelectric output.The increase in ber diameter for the PVDF-SDS-FAQ(LDLK) 3 scaffold is due to the formation of a very ne spiderweb-like mesh (<50 nm diameter) due to the existence of SDS and SAP, which is excluded from the average diameter calculation of common electrospun nanobers.
For the aligned ber scaffold, resultant bers showed uniform morphology, clear alignment, and a slight decrease in the diameter of the bers along with an overall narrower diameter distribution.The degree of directionality of the bers was analyzed as an important function for their potential inuence over cell alignment once seed on the scaffold.To evaluate the ber alignment, derivatives of Fast Fourier Transformations (FFT), color-coded images, and an oval plugin for SEM images were employed.The obtained intensity spectrum of the aligned scaffold showed the peaks related to the main ber Fig. 3 The orientation of fibers was measured on electrospun scaffolds onto a stationary collector and rotating drum (2000 rpm rotating speed).Fast Fourier Transform (FFT) spectra showed a broad distribution of intensities for a stationary collector and two clear peaks for a high-speed rotating collector which are characteristics of highly organized structures.Fiber alignment was significantly increased by using a high-speed rotating collector.
directions at the angles of 108 and 286°(Fig.3).On the other hand, ber alignment was quantied as a coherency value by OrientationJ. 28The coherency values of aligned and random bers are 0.673 and 0.084, respectively.Since a higher value indicates a stronger coherent orientation of bers, the majority of bers are indicated to be aligned in the PVDF-SDS-al scaffold while PVDF-SDS showed disordered ber behavior.
All scaffold conditions exhibited estimated porosity values greater than 90%.However, the aligned scaffold showed a slight decrease in estimated porosity.These results were explained by a ber "packing effect" as the bers aligned, increasing with the anisotropy degree and conrmed from pore size distributions since average pore size increases as the alignment increases. 33ig. 2(j) shows that pore size differed by double for aligned and random ber mesh (PVDF-SDS), with mean pore size of 1.18 ± 0.11 mm 2 and 0.36 ± 0.19 mm 2 , respectively.Considering these attributes, the aligned ber scaffold should promote a higher axon orientation due to its well-aligned bers.However, its larger pore size may impede cell adhesion and proliferation, providing less ber area for neurites and cells to extend and spread. 20Nonetheless, given the considerably "directional" organization of spinal cord tissue, which includes dendrites, axons, and spinal nerves, aligned brous scaffolds have the potential to effectively guide cells and neurites.Despite the considerable variation in the mean ber diameter typically achieved (ranging from <100 nm to 5 mm), brous scaffolds can play a crucial role in this guidance.Notably, there is currently no study that investigates the impact of scaffolds with varying ber diameters on neurite outgrowth when implanted in animal models of SCI. 34arious microscopic characteristics, such as ber diameter and topography, along with macroscopic features like ber orientation, can be manipulated to signicantly alter the physical properties of brous scaffolds.This includes adjusting scaffold porosity, morphology, and architecture.Conversely, the chemical characteristics depend on the inherent properties of the materials used and any chemical modications applied.
A water contact angle test was conducted to assess the wettability of the scaffold surface.This analysis is crucial because a hydrophilic substrate should better mimic the cellular environment, playing a pivotal role in cell attachment, proliferation, and differentiation. 35,36Previous studies have affirmed that alterations in composition can inuence the hydrophilicity of 3D-printed scaffolds. 37Indeed, it has been shown that cell adhesion can be controlled not only by alterations in nanobers diameter and porosity but also surface modication of nanobers. 38,39ther research groups reported that increased wettability can accelerate PVDF degradation and anti-inammatory response. 25,40Fig. 4 indicates the effect of SDS, SAP modication, and ber alignment on scaffolds' wettability.It is observed that scaffolds modied by SAPs (142.50°±1.91°) and SDS (143.75°±3.3°) are more hydrophilic than either PVDF (149.40°± 1.94°) or PVDF-al (147.80°±0.44°) scaffolds.Furthermore, aligned scaffolds show higher hydrophilicity rather than random due to its larger pores.

Electroactive crystalline phases
In its solid state, PVDF is a semi-crystalline polymer which, according to its conformation, can have ve different polar and nonpolar polymorph phases (denoted as: a, b, g, d, and 3). 41The non-polar polymorph a-phase, with anti-parallel stacking of the dipoles, has the lowest energy and is the most stable and favorable phase.However, the b-phase and the g-phase are the most desirable polymorphs as they exhibit strong electrical dipole moments that are responsible for the enhanced piezoelectric activities. 42Gee and colleagues applied the electrospinning method to produce piezoelectric PVDF nanober membranes, and their statistical analysis revealed that the solvent ratio and ow rate are the primary factors affecting the b-phase fraction. 43Additionally, research by Khalifa and colleagues explored the combined impact of electrospinning and nanollers, nding that these elements signicantly enhance the b-phase fraction. 44During electrospinning, the stretching of the electrospun jet in samples containing SDS and SAPs facilitated the formation of the b-phase, while decreasing the a-phase and increasing the band g-phase contents.As a result, the band g-phase are particularly notable for their superior piezoelectric properties. 45ince IR spectroscopy can discriminate between the various crystalline phases of the PVDF, it was used to establish the proportion of polymorph phases of PVDF scaffolds.The vibrational peaks observed at 1071, 1176, and 1397 cm −1 are assigned to bending vibration C-C, swinging vibration CH 2 , and motion vibration CF 2 group of PVDF respectively.The specic spectral regions corresponding to the a, b, and g are between 900 -700 cm −1 and 1250-1200 cm −1 .The vibrational peaks observed at 760 cm −1 are attributed to the non-polar crystalline Although modied scaffolds show improvement in the electroactive phases, the ber-aligned scaffold demonstrated signicant phase modication: the aligned scaffold shows a signicant increase in the b phase (37.7%) while the a phase reduced from 4.9 to 3.2%.It can be due to the extra stretching force applied by high-speed rotation collectors that resulting in further molecular chain conformation and orientation.The high-speed collector inuenced directly the increase of electroactive phases by uniaxially stretched molecular chains, resulting in a higher degree of molecular orientation which promotes the formation of the dipoles. 33The increase of bphase in additive-modied scaffolds is attributed to the ionic bond between positive and negative charges of additives and uorine (−) and hydrogen (+) atoms of PVDF, which would result in the alignment of dipoles. 46In order to determine the relative amorphous-to-crystalline composition of the samples, we studied their melting behavior using DSC.Fig. 5(b) shows rst heating DSC thermograms, giving the melting point (T m ) (endotherm peak), enthalpy of fusion (DH m ), and degree of crystallinity (X c ) of the samples.These values are summarized in Table 2. Thermogram curves of samples demonstrate broad and double endothermal peaks between 160 and 170 °C that are related to the presence of a and b phases, which conrms the FTIR results.According to previous research, DSC is not used to distinguish these two phases, but to calculate the degree of crystallinity of the samples. 47Given the measured degree of crystallinity, the addition of additives and aligned bers doesn't increase the degree of crystallinity.All information from FTIR and DSC is summarized in the bar chart (Fig. 5(d)).It reveals that, by adding SDS and SAPs or fabricating an aligned scaffold, the electroactive phase increased signicantly while the proportion of amorphous and crystal phases remained relatively constant.

Piezoelectric response test
As shown in Fig. 6, through the sound-mechanical strain test all the samples have been determined to have piezoelectric response.These results demonstrate that all electrospun PVDF brous scaffolds are getting electrically poled by mechanical vibration due to piezoelectric properties.All samples have shown a maximum voltage when stimulated between 100 and 500 Hz frequency.The voltage generated by piezoelectric materials is directly proportional to both the rate of strain (how quickly the material deforms) and the d33 component of the material's dielectric tensor.When subjected to low-frequency sound waves, the lm experiences compression and extension at a rate that escalates with increasing frequency.Greater deformation, or the speed at which the lm compresses and recovers in response to external forces, coupled with heightened electroactivity, yields a higher output voltage.However, as the strain rate escalates, the lm becomes unable to adequately respond to deformation, resulting in a diminishing piezoelectric voltage that eventually reaches zero at higher frequencies.The variation in the maximum charge among the samples can be attributed to the correlation between the thickness of the PVDF mesh and its deformability and recovery under mechanical strain, and the generated voltage.The ndings reveal a linear association between mesh thickness and voltage (ESI Table 1).Given the diverse thicknesses and volumetric densities of the meshes, we approached this sound test experiment as a qualitative test of piezoelectricity.

In vitro assays
In vitro investigations to test cell differentiation and cell behavior were carried on the electrospun PVDF-based nanobrous mats. 48ble 2 Melting temperature (T m ), melting enthalpy (DH m ), and degree of crystallinity (X c ) of electrospun PVDF scaffolds  The morphology of differentiated murine neural stem cells (mNSCs) was studied on ve different types of electrospun nanobrous mats (PVDF, PVDF-SDS, PVDF-SDS-al, PVDF-FAQ(LDLK) 3 and PVDF-SDS-FAQ(LDLK) 3 ).The differentiation of NSCs progeny at 7 DIV was assessed by staining neurons with the bIII-TUB marker, astrocytes with GFAP, and oligodendrocytes with GALC/O4.The hNSCs were distributed uniformly aer 7 DIV in culture on electrospun PVDF scaffolds and showed a typical polygonal morphology with long cytoplasmic extensions and clearly visible cell-cell contacts.Diverse cell morphologies were observed upon cultivation on PVDF ber surfaces (Fig. 7).Cells cultured on Cultrex displayed a robust bidirectional alignment, predominantly adopting a bipolar morphology (Fig. 7).In contrast, NSCs cultured on PVDF showcased a polygonal structure, demonstrating a pronounced inclination to align along the material bers (Fig. 7), as highlighted by elongated cytoplasmic extensions.Glial cells exhibited a stretched and radiant network of dendrites, while neuronal cells displayed elongation along a distinct axis.
Based on the data presented in Fig. 7, bIII-TUB served as a marker indicating the differentiation into neurons of NSCs.Upon comparison with Cultrex, NSCs efficiently differentiate into neuronal cells when cultured on PVDF-SDS-FAQ(LDLK) 3 .This could be due to the modication of the surface chemistry of the scaffold by the addition of both SDS and SAPs, which resulted in increased hydrophilicity, functionalization with the FAQ pro-neuronal motif 49 and subsequently cell attachment.In the context of the GFAP marker, all the samples exhibited comparable results to Cultrex, with no signicant differences observed in the immunostaining of GFAP.As for the length of axons, a statistically signicant difference was observed between electrospun PVDF scaffolds and the control, Cultrex.Additionally, GFAP staining was employed for the analysis of the soma's area.In electrospun PVDF scaffolds treated with SDS, the soma's area of GFAP-positive cells exhibited a signicant difference compared to Cultrex.It is crucial to emphasize that the size, distribution, and interconnectivity of pores play a pivotal role in governing the diffusion rates of nutrients and the removal of waste.A substrate with an optimal pore size not only facilitates cell seeding and penetration but also enhances oxygen diffusion and distribution within the scaffolds.Furthermore, pore size signicantly inuences cell adhesion, intercellular interactions, and cell spreading.Neurons have the ability to sense the scaffold surface, and focal adhesions play a regulatory role in signaling complexes and integrin function.This triggers a signaling cascade that promotes cell proliferation and differentiation. 50,51Moreover, to corroborate these data, cell viability studies were performed using MTS assay.Cell proliferation and viability were assessed with an MTS assay (ESI Fig. 3).Cells seeded on gold standard Cultrex produced comparable values when compared to treated samples.In the case of the aligned scaffold, we note that the reduction in axon length and soma area size was predictable due to the increasing pore size and the corresponding reduction in the area available for cell spreading.The effectiveness of PVDF meshes as a potential therapy needs to be assessed, especially concerning neural connectivity.Understanding how these scaffolds impact neural connectivity is essential for evaluating their potential to promote cell proliferation and differentiation in the context of neural therapy.On the other hand, as shown in Fig. 8, neuronal cells exhibited irregular morphology on the prepared random PVDF-SDS.In comparison, the growth of neuronal cells on the aligned scaffold (PVDF-SDS-al) is orientated at 85°. Cell orientation was quantied as a coherency value of aligned and random scaffolds that are 0.431 and 0.065, respectively, which proved a stronger coherent orientation of bers for cell growth on aligned scaffolds.These results imply that using an aligned scaffold guides cell growth.
Ensuring continuity in the transmission of information is crucial for regaining lost locomotor performance.Therefore, the quantitative evaluation of the evolution of neural processes' directionality over time was essential. 52The materials and methods provided a comprehensive description, illustrating a side-by-side comparison of the mean directionality in Cultrex (used as control) and electrospun PVDF meshes.
Ultimately, the percentage of oligodendrocytes was assessed, as indicated in Fig. 9, employing GalC-O4 markers.In this instance, notable differences were observed among Cultrex and electrospun scaffolds in terms of oligodendroglial differentiation suggesting a signicant impact of scaffold composition on NSCs differentiation.
Specically, Cultrex displayed the lowest oligodendrocyte percentage compared to PVDF-SDS, PVDF-SDS-al, and PVDF-SDS-FAQ(LDLK) 3 highlighting the importance of scaffold material in guiding cellular differentiation.One relevant paper by Thorrez et al., 53 discusses the differentiation of NSCs into oligodendrocytes on poly(L-lactide) (PLLA) nanober scaffolds.While the scaffold material differs from the ones mentioned in the excerpt, the underlying principles of scaffold inuence on oligodendroglial differentiation are likely to be relevant.Another noteworthy distinction in oligodendrocyte percentage was observed between PVDF and PVDF-SDS.It is worth noting that in samples with the highest oligodendrocyte percentage, SDS was present.This suggests that the addition of SDS (and overall hydrophilicity of the scaffold) may contribute to the production of bers more conducive to the oligodendroglial differentiation of NSCs.This observation aligns with previous research indicating the importance of scaffold properties, such as hydrophilicity, in directing cellular behavior and differentiation.
Various tissues necessitate specic microenvironments to support fundamental processes such as cell-cell interaction, migration, proliferation, differentiation, and regeneration.In particular, materials derived from PVDF have proven effective as substrates for neuron attachment, growth, and differentiation, with their properties inuenced by electrical activity. 33n the eld of neural tissue engineering, electrospun brous mats with aligned bers offer a signicant advantage over scaffolds featuring randomly oriented bers.This advantage is attributed to the spatial guidance provided by highly aligned bers, which facilitates neurite outgrowth and axonal elongation along specic directions in the attempt to mimic the native nervous tissue, like in the spinal cord.
NSCs, already proved to be crucial for nerve function and repair in vivo, 54 were chosen to evaluate the potential of electrospun PVDF scaffolds in vitro as potential neural cell carriers in future therapies for nervous regeneration.The impact of scaffold morphology, including factors such as ber size, alignment, and porosity as well as structural properties like crystallinity and wettability, is widely acknowledged for its inuence on cell morphology, growth, and differentiation. 55ins et al. conducted an analysis on the inuence of PVDF ber alignment on both undifferentiated monkey NSCs and the subsequent differentiation into neuronal and glial cells. 33The study revealed that while the growth patterns of undifferentiated stem cells and glial cells remained unaffected by ber alignment, differentiated neuronal cells exhibited an elongated morphology specically on aligned bers.
In comparison to previous studies like Lins et al., our work likely contributes to the scientic community by introducing novel elements such as the use of specic additives like SAPs and SDS in the fabrication of PVDF scaffolds.These additives may have been incorporated to modify scaffold properties such as surface wettability and surface chemistry, which could in turn inuence cell-scaffold interactions and ultimately cellular behavior.Therefore, our work extends beyond simply assessing the impact of scaffold morphology on NSCs to exploring the effects of scaffold composition and surface properties on NSC behavior, which adds valuable insights to the eld of neural tissue engineering and regeneration.

Conclusion
This study successfully identied and characterized electrospun PVDF scaffolds conducive to nervous cell growth, highlighting their potential for spinal cord injury regeneration therapies.We explored the effects of incorporating SDS and SAPs additives, as well as utilizing different electrospinning techniques (static target and rotating drum collector), on ber morphology and electroactive phase content.
Our ndings revealed that the addition of SDS and SAPs, as an anionic surfactant and bioactive agent respectively, combined with the use of a rotating collector, resulted in uniform, ner bers with higher alignment and increased hydrophilicity (notably in PVDF-SDS and PVDF-FAQ(LDLK)3 samples).DSC and IR spectroscopy data indicated a signicant increase in electroactive phase content with the inclusion of these additives.Furthermore, the rotating collector was particularly effective in inducing b-phase generation in modi-ed PVDF scaffolds.In terms of biological performance, hNSCs seeded on these electrospun nanobrous scaffolds exhibited satisfactory proliferation, viability, and differentiation compared to a gold standard.The scaffolds supported cell differentiation into the three main neural phenotypes and promoted notable cell sprouting.The tested SAP demonstrated benecial effects, suggesting that other functionalized peptide molecules could further enhance scaffold multifunctionality, thereby boosting host nervous regeneration and transplanted cell engrament.Despite the challenge of larger pores potentially hampering cell survival and axon length, aligned scaffolds were shown to promote a higher degree of cell orientation due to the nano-and microber alignment.Ultimately, our results pave the way for the development of electroactive, biomimetic brous scaffolds with tailored architectures, offering promising applications in neural tissue engineering.

Paper
RSC Advances

Fig. 1
Fig. 1 Schematic of piezoelectric response test.Stimulated by different frequencies.

Fig. 2
Fig. 2 SEM micrographs along with diameter distribution of electrospun PVDF scaffolds with and without additives: (a) electrospun PVDF scaffold fabricated by DMF/acetone (60 : 40) solution shows non-uniform fibers including spindle shape beads and fibers with the average diameter of 444.4 ± 15.6 nm, (b) PVDF-SDS, electrospun scaffolds with more uniformity of fibers after adding a surfactant and average diameter of 300.6 ± 13.1 nm, (c) electrospun PVDF-SDS-al scaffold comprises defect-free fibers with highly-alignment shows thinner fibers about 328.1 ± 11.1 nm (d) electrospun PVDF made of 100% DMF solution reveals round shape beads and very thin fibers, (e) electrospun PVDF-SDS scaffold made of 100% DMF solution contains fewer beads and more fibrous content (f) PVDF-FAQ(LDLK) 3 electrospun scaffold including SAPs reveals defect-free and uniform fibers with average diameter of 361.7 ± 14.7 nm, (g) PVDF-SDS-FAQ(LDLK) 3 electrospun scaffold contains SDS and SAPs with very uniform fibers and lowest bead content but higher average diameter 572.3 ± 14.8 nm, (h) Violin plot of the diameter distribution of electrospun scaffolds, (i) porosity and bead content plot, (j) pore size.Scale bars present 20 mm.

Fig. 4
Fig.4The water contact angle for electrospun scaffolds with different statuses.The line graph shows the reduction of contact angle after adding SDS or SAPs from about 150°to below 145°.

Fig. 5
Fig. 5 (a) FTIR spectra along with the specific absorption peaks corresponding to the a, b, and g to measure phase content, (b) DSC thermogram showing melting peaks to measure the degree of crystallinity of scaffolds, (c) phase content measured by the three mathematical resolution enhancement methods of Fourier self-deconvolution, second derivative analysis, and band curve-fitting of FTIR spectra.(d) Bar charts representing percentages of each phase and the electroactive phase are determined by merging phase content (FTIR) and degree of crystallinity (DSC) results.The orange box indicates the overall electroactive phase content.Data are represented as average ± SEM (N = 3).Statistical analysis: oneway ANOVA followed by Tukey multiple comparison tests.Statistical analysis shows significant differences between conditions (*p # 0.05, **p # 0.01, and ***p # 0.001).

Fig. 6
Fig. 6 Piezoelectric response test.Stimulated PVDF scaffolds under different frequencies.All samples were subjected to electrical polarization, resulting in the subsequent generation of voltage through frequency vibration stimulation.

Fig. 7
Fig. 7 (a) Fluorescence microscopic images were captured of NSCs on PVDF mats, as well as on Cultrex (control), at the conclusion of the 7 days differentiation period.b-Tubulin (bIII-TUB) was stained in green, glial fibrillary acidic protein (GFAP) in red, and cell nuclei in blue.(b) Percentages of bIII-TUB cells.(c) Percentages of GFAP-positive cells.d) Axon's length is expressed in mm.(e) Soma's area is expressed in mm 2 .Scale bar presents 50 mm.Statistical analysis: one-way ANOVA followed by Tukey multiple comparison test.Statistical analysis shows significant differences between conditions (*p # 0.05, **p # 0.01, and ***p # 0.001).

Fig. 8
Fig. 8 Nanofibers alignment regulates neuronal cell orientation.Fluorescence images show a disordered spread of cells on the random scaffolds (PVDF-SDS).In contrast, cell growth on the aligned nanofibers (PVDF-SDS-al) shows an ordered pattern.Entire visual outcomes were proved by alignment quantification, two peaks at 85 and 265°for PVDF-SDS-al scaffold, and coherency values (much higher coherency value for PVDF-SDS-al rather than PVDF-SDS random scaffold.Scale bar presents 50 mm.

Fig. 9
Fig. 9 (a) Fluorescence microscopic images of hNSCs on PVDF mats, as well as Cultrex (control) at the end of the 7 days differentiation period.GALC/O4 were stained in red and nuclei in blue.(b) Percentages of GALC/O4 positive cells.(c) Numbers of cell nuclei counterstained with DAPI.Scale bar presents 50 mm.Statistical analysis: one-way ANOVA followed by Tukey multiple comparison test.Statistical analysis shows significant differences between conditions (*p # 0.05, **p # 0.01, and ***p # 0.001).